Since the inception of this project we have determined the complete structures of the two groups of phosphoproteins in human saliva which inhibit precipitation of calcium phosphate. These proteins are represented by statherin, a tyrosine rich acidic phosphoprotein, and the anionic proline rich protein, PRP-4, the most potent of the PRP's. During the second year of the project we have also obtained a tentative complete sequence of PRP-2, a 150 residue PRP, and have localized the active site of all the human PRP's to the amino terminal 30 residue tryptic peptide and the 38 amino terminal tryptic peptide in Macaca arctoides. In addition the statherin-like peptide in M. arctoides has been purified to homogeneity. With respect to statherin of human origin, we have been able to demonstrate that the NH2-terminal hexapeptide, containing the two phosphoserine residues, contains the part of the sequence required for phase-transformation inhibition but not for spontaneous precipitation. In fact the hexapeptide is 4 fold as active as intact statherin in this assay system. Similarly other N-terminal peptides have been purified following enzymatic digestion; these include the N-terminal heptapeptide, nonapeptide, decapeptide and N-terminal 18-amino acid peptide. With increasing C-terminal extension, phase transformation inhibition decreases. Research performed this last year included solid phase peptide synthesis of the phosphoserine dipeptide, NH2-Pser-Pser-COOH, the N-terminal pentapeptide and the N-terminal decapeptide of statherin. These peptides are also being synthesized via the classical solution approach. Initial results indicate, suprisingly, that the phosphoserine dipeptide is inactive but that the penta- and decapeptide have considerable activity suggesting that the peptide backbone contain part of the statherin structure is required for biological activity. These peptide analogs will be studied by 31P NMR to determine the calcium binding site and to learn the conformation of these interesting macromolecules. Finally in collaboration with Frederic Coe, at Michael Reese Hospital in Chicago, we are beginning to study the structure of an inhibitor of calcium oxalate precipitation in human urine.